Organization of the ferritin genes in Drosophila melanogaster

The organization of two closely clustered genes, Fer1HCH and Fer2LCH, encoding the heavy-chain homolog (HCH) and the light-chain homolog (LCH) subunits of Drosophila melanogaster ferritin are reported here. The 5019-bp sequence of the cluster was assembled from genomic fragments obtained by polymerase chain reaction (PCR) amplification of genomic DNA and from sequences obtained from the Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org). These genes, located at position 99F1, have different exon-intron structures (Fer1HCH has three introns and Fer2LCH has two introns) and are divergently transcribed. Computer analysis of the possibly shared promoter regions revealed the presence of putative metal regulatory elements (MREs), a finding consistent with the upregulation of these genes by iron, and putative NF-kappaB-like binding sites. The structure of two other invertebrate ferritin genes, from the nematode Caenorhabditis elegans (located on chromosomes I and V), was also analyzed. Both nematode genes have two introns, lack iron-responsive elements (IREs), and encode ferritin subunits similar to vertebrate H chains. These findings, along with comparisons of ferritin genes from invertebrates, vertebrates, and plants, suggest that the specialization of ferritin H and L type chains, the complex exon-intron organization of plant and vertebrate genes, and the use of the IRE/iron regulatory protein (IRP) mechanism for regulation of ferritin synthesis are recent evolutionary acquisitions.     

Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism

The Irr protein from the bacterium Bradyrhizobium japonicum is expressed under iron limitation to mediate iron control of haem biosynthesis. The regulatory input to Irr is the status of haem and its precursors iron and protoporphyrin at the site of haem synthesis. Here, we show that Irr controls the expression of iron transport genes and many other iron-regulated genes not directly involved in haem synthesis. Irr is both a positive and negative effector of gene expression, and in at least some cases the control is direct. Loss of normal iron responsiveness of those genes in an irr mutant, as well as a lower total cellular iron content, suggests that Irr is required for the correct perception of the cellular iron status. Degradation of Irr in iron replete cells requires haem. Accordingly, control of Irr-regulated genes by iron was aberrant in a haem-defective strain, and iron replete mutant cells behave as if they are iron-limited. In addition, the haem mutant had an abnormally high cellular iron content. The findings indicate that B. japonicum senses iron via the status of haem biosynthesis in an Irr-dependent manner to regulate iron homeostasis and metabolism.     

Functional conservation of clock-related genes in flowering plants: overexpression and RNA interference analyses of the circadian rhythm in the monocotyledon Lemna gibba

Circadian rhythms are found in organisms from cyanobacteria to plants and animals. In flowering plants, the circadian clock is involved in the regulation of various physiological phenomena, including growth, leaf movement, stomata opening, and floral transitions. Molecular mechanisms underlying the circadian clock have been identified using Arabidopsis (Arabidopsis thaliana); the functions and genetic networks of a number of clock-related genes, including CIRCADIAN CLOCK ASSOCIATED1, LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1, GIGANTEA (GI), and EARLY FLOWERING3 (ELF3), have been analyzed. The degree to which clock systems are conserved among flowering plants, however, is still unclear. We previously isolated homologs for Arabidopsis clock-related genes from monocotyledon Lemna plants. Here, we report the physiological roles of these Lemna gibba genes (LgLHYH1, LgLHYH2, LgGIH1, and LgELF3H1) in the circadian system. We studied the effects of overexpression and RNA interference (RNAi) of these genes on the rhythmic expression of morning- and evening-specific reporters. Overexpression of each gene disrupted the rhythmicity of either or both reporters, suggesting that these four homologs can be involved in the circadian system. RNAi of each of the genes except LgLHYH2 affected the bioluminescence rhythms of both reporters. These results indicated that these homologs are involved in the circadian system of Lemna plants and that the structure of the circadian clock is likely to be conserved between monocotyledons and dicotyledons. Interestingly, RNAi of LgGIH1 almost completely abolished the circadian rhythm; because this effect appeared to be much stronger than the phenotype observed in an Arabidopsis gi loss-of-function mutant, the precise role of each clock gene may have diverged in the clock systems of Lemna and Arabidopsis.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Circadian clock regulates photoperiodic responses governed by distinct output pathways in the bean bug,Riptortus pedestris

Effects of RNA interference (RNAi) targeted against circadian clock genes on two distinct types of photoperiodic responses – ovarian development and lipid accumulation – were investigated in a bean bug Riptortus pedestris, to explore which physiological process in the photoperiodic response involved the circadian clock. Ovarian development and lipid accumulation are known to be regulated by distinct output pathways. Control insects showed clear photoperiodic responses; i.e. induction of ovarian development and suppression of lipid accumulation under long-day conditions, whereas opposite characteristics under short-day conditions. We found that RNAi directed against period, a negative element of the circadian clock, produced a long-day effect for both the ovarian development and lipid accumulation, while RNAi directed against Clock, a positive element of the circadian clock, produced a short-day effect for both, irrespective of photoperiod. These results indicate that the circadian clock comprised of these genes regulates a process governing both distinct photoperiodic responses.     

Casein kinase 2, circadian clocks, and the flight from mutagenic light

Circadian clocks play a fundamental role in biology and disease. Much has been learned about the molecular underpinnings of these biological clocks from genetic studies in model organisms, such as the fruit fly, Drosophila melanogaster. Here we review the literature from our lab and others that establish a role for the protein kinase CK2 in Drosophila clock timing. Among the clock genes described thus far, CK2 is unique in its involvement in plant, fungal, as well as animal circadian clocks. We propose that this reflects an ancient, conserved function for CK2 in circadian clocks. CK2 and other clock genes have been implicated in cellular responses to DNA damage, particularly those induced by ultraviolet (UV) light. The finding of a dual function of CK2 in clocks and in UV responses supports the notion that clocks evolved to assist organisms in avoiding the mutagenic effects of daily sunlight.     

Genome-wide transcriptional orchestration of circadian rhythms in Drosophila

Circadian rhythms govern the behavior, physiology, and metabolism of living organisms. Recent studies have revealed the role of several genes in the clock mechanism both in Drosophila and in mammals. To study how gene expression is globally regulated by the clock mechanism, we used a high density oligonucleotide probe array (GeneChip) to profile gene expression patterns in Drosophila under light-dark and constant dark conditions. We found 712 genes showing a daily fluctuation in mRNA levels under light-dark conditions, and among these the expression of 115 genes was still cycling in constant darkness, i.e. under free-running conditions. Unexpectedly the expression of a large number of genes cycled exclusively under constant darkness. We found that cycling in most of these genes was lost in the arrhythmic Clock (Clk) mutant under light-dark conditions. Expression of periodically regulated genes is coordinated locally on chromosomes where small clusters of genes are regulated jointly. Our findings reveal that many genes involved in diverse functions are under circadian control and reveal the complexity of circadian gene expression in Drosophila.     

The circadian clock, light, and cryptochrome regulate feeding and metabolism in Drosophila

Recent studies in mammals have demonstrated a central role for the circadian clock in maintaining metabolic homeostasis. In spite of these advances, however, little is known about how these complex pathways are coordinated. Here, we show that fundamental aspects of the circadian control of metabolism are conserved in the fruit fly Drosophila. We assay feeding behavior and basic metabolite levels in individual flies and show that, like mammals, Drosophila display a rapid increase in circulating sugar following a meal, which is subsequently stored in the form of glycogen. These daily rhythms in carbohydrate levels are disrupted in clock mutants, demonstrating a critical role for the circadian clock in the postprandial response to feeding. We also show that basic metabolite levels are coordinated in a clock-dependent manner and that clock function is required to maintain lipid homeostasis. By examining feeding behavior, we show that flies feed primarily during the first 4 hours of the day and that light suppresses a late day feeding bout through the cryptochrome photoreceptor. These studies demonstrate that central aspects of feeding and metabolism are dependent on the circadian clock in Drosophila. Our work also uncovers novel roles for light and cryptochrome on both feeding behavior and metabolism.     

Male reproduction is affected by RNA interference of period and timeless in the desert locust Schistocerca gregaria

In all living organisms, behavior, metabolism and physiology are under the regulation of a circadian clock. The molecular machinery of this clock has been conserved throughout the animal kingdom. Besides regulating the circadian timing of a variety of processes through a central oscillating mechanism in the brain, these circadian clock genes were found to have a function in peripheral tissues in different insects. Here, we provide evidence that the circadian clock genes period (per) and timeless (tim) have a role in the male locust reproduction. A knockdown of either of the two genes has no effect on male sexual maturation or behavior, but progeny output in their untreated female copulation partners is affected. Indeed, the fertilization rates of the eggs are lower for females with a per or tim RNAi copulation partner as compared to the eggs deposited by females that mated with a control male. As the sperm content of the seminal vesicles is higher in per or tim knockdown males, we suggest that this phenotype could be caused by a disturbance of the circadian regulated sperm transfer in the male reproductive organs, or an insufficient maturation of the sperm after release from the testes.     

Functional analysis of genes differentially expressed in the Drosophila wing disc: role of transcripts enriched in the wing region

Differential gene expression is the major mechanism underlying the development of specific body regions. Here we assessed the role of genes differentially expressed in the Drosophila wing imaginal disc, which gives rise to two distinct adult structures: the body wall and the wing. Reverse genetics was used to test the function of uncharacterized genes first identified in a microarray screen as having high levels of expression in the presumptive wing. Such genes could participate in elaborating the specific morphological characteristics of the wing. The activity of the genes was modulated using misexpression and RNAi-mediated silencing. Misexpression of eight of nine genes tested caused phenotypes. Of 12 genes tested, 10 showed effective silencing with RNAi transgenes, but only 3 of these had resulting phenotypes. The wing phenotypes resulting from RNAi suggest that CG8780 is involved in patterning the veins in the proximal region of the wing blade and that CG17278 and CG30069 are required for adhesion of wing surfaces. Venation and apposition of the wing surfaces are processes specific to wing development providing a correlation between the expression and function of these genes. The results show that a combination of expression profiling and tissue-specific gene silencing has the potential to identify new genes involved in wing development and hence to contribute to our understanding of this process. However, there are both technical and biological limitations to this approach, including the efficacy of RNAi and the role that gene redundancy may play in masking phenotypes.