Takamichi Muramatsu

Fluorescent Differential Display Identifies Circadian Clock-Regulated Genes in Arabidopsis thaliana

Circadian rhythms in gene expression were first observed in plants more than 13 years ago, but the underlying mechanism controlling rhythmic gene expression is still not understood. The isolation of novel circadian clock-controlled genes (ccgs) is likely to provide new tools for studying circadian rhythms. Fluorescent differential display (FDD) was used to screen Arabidopsis thaliana mRNAs for cycling transcripts. Seventy PCR primer pairs were screened, and 17 different cycling bands were observed out of an estimated 10,500 bands screened. The identities of 10 bands were determined, and the rhythmic gene expression was confirmed using northern blot analysis. The 10 cycling bands represent 7 different genes, 6 of which are present in the databases and 1 that does not match anything in current databases. The rhythmic expression of the 7 genes is composed of four distinct phases of clock regulation. The results demonstrate that FDD can be used to isolate ccgs. The genes identified in this screen range from known A. thaliana ccgs, as well as genes shown to be clock controlled in other plant species, to a novel gene that may encode a pioneer protein. Further study of these ccgs is likely to increase our understanding of circadian-regulated gene expression.

Receptor activator of NF-κB ligand-dependent expression of caveolin-1 in osteoclast precursors, and high dependency of osteoclastogenesis on exogenous lipoprotein

Although extensive studies have done much to clarify the molecular mechanisms of osteoclastogenesis during the last ten years, there may still be unknown molecules associated with osteoclast differentiation. Thus, we used fluorescent differential display to screen for genes whose expression is induced by receptor activator of NF-κB ligand (RANKL), a crucial molecule for osteoclast formation. We identified caveolin-1 (Cav-1) as a RANKL-induced gene. Cav-1 is a major structural protein of caveolae and lipid rafts, cholesterol-enriched microdomains in the plasma membrane (PM). The RANKL-induced Cav-1 was immediately conveyed to lipid rafts. Conversely, expression of flotillin-1 (Flot-1), another scaffolding protein of lipid rafts, was reduced during osteoclastogenesis, indicating conversion of Flot-1-predominant rafts into Cav-1-enriched rafts. However, in vitro osteoclastogenesis of precursor cells from Cav-1-null mice was comparable to that of wild-type mice, while Cav-2 expression in the knockout osteoclasts was maintained. Conversely, Cav-2 gene silencing in Cav-1-null osteoclast precursors using siRNA for Cav-2 increased osteoclast formation, suggesting that the Cav-1/Cav-2 complex may act as a negative regulator for osteoclastogenesis. On the other hand, destruction of lipid rafts by removal of cholesterol from the PM by methyl-ß-cyclodextrin (MCD) treatment caused disordered signal transductions for osteoclastogenesis, such as hyperactivation of Erk1/2 and insensitivity of Akt to RANKL stimulus. The abnormal signaling was reproduced by deleting exogenous lipoproteins from the culture medium, which also resulted in reduced osteoclast formation. In addition, the deletion caused delayed expression of nuclear factor of activated T cells c1 (NFATc1), and depressed its activation in the cytosol and inhibited its translocation into nuclei. Simultaneously, the deletion reduced the level of FcRγ, a trigger protein for initiating the calcium signaling needed to activate NFATc1, and decreased Cav-1 in lipid rafts. These findings indicate that the molecular mechanisms of osteoclastogenesis are highly dependent on extracellular lipoprotein and the integrity of lipid rafts, and suggest possible involvement of cholesterol.

OVER-EXPRESSION OF A NEW PHOTO-ACTIVE HALORHODOPSIN IN HALOBACTERIUM SALINARIUM

The gene of haloopsin (hop) from halobacterial strain shark was cloned and its nucleotide sequence was determined. The deduced amino acid sequence of shark halorhodopsin (HR) showed that its homology with halobium HR was 62%. The gene product seems to be HR having several positively charged residues that are conserved in all known HRs. The gene encoding shark hop as well as that encoding halobium hop were successfully expressed in Halobacterium salinarium (halobium) by using a plasmid shuttle vector containing the bacterioopsin (bop) promoter. The expression level of shark HR is almost the same as that for halobium HR with the same shuttle vector containing the bop promoter. Under the physiological conditions, the anion pumping activity of the shark HR expressed in H. salinarium was almost the same as that for halobium HR; however, the anion selectivity and half-maximal anion transport were different. Furthermore, its absorption maximum in the absence of chloride shifted to approx. 596 nm in contrast to that for halobium HR. The half-lifetimes of HR520 formation for shark HR and halobium HR were almost the same; however, the half-lifetime of its decay was approx. 6-times faster for shark HR than it was for halobium HR at a high chloride concentration (1000 mM). Even at a low chloride concentration (50 mM), HR520 and HR640 intermediates could be detected for shark HR, and the half-lifetime of HR640 decay was found to be approx. 25 ms. In the presence of nitrate, the half-lifetime of HR565 recovery for shark HR was approx. 10-times slower than that for halobium HR. Some of amino acid substitutions between shark HR and halobium HR may affect the anion selectivity and the photoreaction of HR.