Koichiro Kako

Highly conserved Drosophila ananassae timeless gene functions as a clock component in Drosophila melanogaster

The behavior and physiology of Drosophila are subject to rhythms that are controlled by the circadian clock genes, period, timeless, clock and cycle, all of which are thought to participate in central pacemaker control. The molecular mechanism of rhythm in Drosophila has been studied in detail. However, rhythm and clock genes have mostly been analyzed in Drosophila melanogaster. To confirm whether the tim gene exists and works as a clock component in other Drosophila species, we cloned a tim homolog from Drosophila ananassae that shared 85.9% similarity with Drosophila melanogaster tim at the amino acid level. In addition, the PER interaction domains and NLS were highly conserved. Introduction of the D. ananassae tim homolog rescued the rhythm of the locomotor activity of about 44% of a population of D. melanogaster tim(01) flies. At the molecular level, hs-tim introduced not only TIM but PER oscillation in transgenic flies. These results indicate that the tim gene in D. ananassae functions as a component of the circadian clock in D. melanogaster.

Genomic structure of mouse copper chaperone, COX17.

Cox17p was first cloned as a cytoplasmic copper chaperone from yeast mutant and recent works suggested the existence of mammalian homologues. Previous report has shown that a gel filtration fraction of heart extract containing porcine Coxl7p peptide promoted the survival of NIH3T3 fibroblast cells. In the present study, we first cloned DNA fragments of the mouse COX17 gene. The mouse COX17 spans ∼6kb and consists of three exons. It was mapped to the center of chromosome 16, using a radiation hybrid-mapping panel. The major transcription start site is 80 bp upstream of the ATG initiation codon as determined by rapid amplification of cDNA ends (5′-RACE) analysis. Two potential polyadenylation sites are 3233 and 3293 bp downstream of the termination codon, respectively. Transient transfection of reporter plasmids containing portions of the mouse COX17 5′-flanking region into AtT-20 and NIH3T3 cells allowed the localization of the essential promoter to a 0.8 kb region upstream of the transcription starting site. Furthermore, the transfected luciferase activity was much higher in AtT-20 than NIH3T3. According to sequence analysis of the –0.8 kb 5′-flanking region, GC rich segments including consensus sequences for binding of the transcription factor Sp1, but no The TATAICAAT boxes, exist in the region of the transcription start site. Besides the GC box, binding sites for NRF-1 and 2 known as specific transcription factors for COX subunits are also localized around the transcription starting site.

Detection of PERIOD/PAS-binding proteins from rat brain nuclear extracts by affinity chromatography

In Drosophila, period and timeless genes products (dPER and dTIM) have been identified to act as circadian clock components in heterodimeric form. The formation of dPER-dTIM complex is based on the interaction between dPERs PAS domain and dTIMs PAS binding domain. As an initial step to understand the molecular mechanism of mammalian circadian clock, we screened His-tagged dPER/PAS binding proteins from rat brain nuclear extracts, using Ni2+-NTA affinity chromatography. As a result of screening, we identified two proteins (192 and 180 kDa), which specifically bound to His-dPER/PAS in rat brain extracts. Such proteins might be a candidate of rat timeless proteins.

Effect of Deficiency of Cytochrome C Oxidase Assembly Peptide Cox17p on Mitochondrial Functions and Respiratory-Chain in Mice

A gel filtration fraction of porcine heart extract promoted the survival of NIH3T3 fibroblast cells in serum-free medium [1]. In addition to the porcine peptide [1], human, rat [2], and mouse [2] Coxl7p homologues have been discovered to date (Figure 1). Although it has been believed that Cox17p guides Cu to mitochondria for insertion into cytochrome c oxidase (COX) in yeast, its physiological role in mammals is unknown. Recently, we have found that expression levels of Cox17p mRNA are high in mouse heart, kidney, brain and some endocrine cell lines, but quite low in small intestine, liver and some fibroblast cell lines [2]. Using the GST-fusion protein technology, we show that the GST-mCoxl7p (mouse Cox17p) binds copper at the putative metal-binding motif (-KPCCXC-) in vitro [3]. We have isolated COX17 genomic DNA and shown that transcription factors Spl and NRF-1 drive the basal transcription of this gene [4]. Since our goal is to determine the physiological function of this novel peptide, we hypothesized that mammalian Cox17p peptide delivers intracellular Cu to the cuproenzymes, such as COX.

Diurnal regulation of a DNA binding protein to the period repeat sequence in the SCN nuclear extract of rat brain

We previously reported the mammalian period repeat mRNA fluctuates during circadian time in the rat suprachiasmatic nucleus (SCN) which is considered to be a clock pacemaker in mammalian brain. Presently we discovered a period repeat sequence (PR) DNA-binding protein in the rat SCN nuclear extract. In the SCN, the binding activity of PR DNA-binding protein to (ACAGGC)3 was most highest during the late day and most lowest during the late night by electro-mobility shift assay (EMSA). In the cortex nuclear extract, the binding of PR DNA-binding protein did not show a significant variation during a day. This is the first report to show the existence of diurnal regulated PR DNA-binding protein in the SCN.

2106 Circadian expression rhythm of serotonin N-acetyltransferase gene in the mammalian retina

KATSUHIKO SAKAMOTO’, TOMOKO NIKI’, SATORU SUZUKI’, TOSHIYUKI HAMADA’, KAZUMASA HORIKAWA’, MICHIKO OOTOM12, KOICHIRO KAKO’, NORIO ISHIDA’ We examined the temporal expression pattern of serotonin N-acetyltransferase (NAT; the rate-limiting enzyme in melatonin synthesis) gene in the rat retina. Retina NAT mRNA exhibited rhythmic expression synchronizing to a daily light-dark cycle, with peak levels at night. The NAT mRNA rhythm persisted in constant darkness, which suggests that the NAT gene expression in the mammalian retina was controlled by an endogenous clock.