Previous cell cycle studies have been based on cell-nuclear proliferation only. conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed. B homologs, CDC5, SCH9, DSK2 and ZPR1. Proteins involved in DNA replication include histones, some checkpoint kinases and some proteins regulating DNA damage and repair. Many groups of genes related to translation and other metabolic processes are also cyclic in all three organisms.10 In or tobacco BY-2, mitochondrial and plastid Rabbit polyclonal to ARMC8 divisions were not shown. Mammalian, herb and yeast cells contain many organelles whose divisions occur at random, cannot be synchronized and have designs that are very diverse and complicated.13 Therefore, genes related to such organelles are not reflected in microarray analyses of the cell cycle in higher organisms. In previous cell cycle studies, the analysis has been based on nuclear proliferation only. Eukaryotic cells, however, have double-membrane-bound organelles, such as the nucleus, mitochondria and plastids, and single-membrane-bound organelle such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferation is very important for cell functions, as well as differentiation and cell division. However, you will find few studies that have investigated the organelle proliferation cycle. The unicellular red alga has advantages for investigating organelle proliferation as it has a minimum set of organelles,14C16 and organelle division can be synchronized by a light/dark cycle.17 The mitochondrial and plastid division requires the FtsZ,18,19 the Dynamin20,21 and the MD/PD rings.22,23 Northern blot analysis has shown that each transcriptional level of for mitochondrial division, 1187595-84-1 and for the plastid division, has a peak per cell cycle before each division.18 Moreover, nuclear, mitochondrial and plastid genomes of have completely been sequenced.24C27 1187595-84-1 As the nucleus genome 4775 ORF’s coding protein 1187595-84-1 includes 27 introns only, newly designed proteins by alternative splicing are few; therefore, the functioning protein is directly identified as the same gene. Furthermore, most ORFs do not have paralogues.24 We thought that novel organelle division-related genes like could be found by genome-wide transcriptome of the cell cycle. 2.?Materials and methods 2.1. Synchronous culture and fluorescence microscopy 10D-14 were synchronized according to the method discussed in Suzuki et al.17 Cells were cultured in 2 Allen’s medium at pH 2.3. Flasks were shaken under continuous light (40 W/m2) at 42C. The cells were sub-cultured to <107 cells/mL, and then synchronized by subjecting them to a 12 h light/12 h dark cycle at 42C while the medium was aerated. For the observation of DNA, the cells were fixed in 1% glutaraldehyde diluted with 2 Allen's medium and stained with 1 g/mL DAPI (4,6-diamidino-2-phenylindole phosphate). Images were viewed using an epifluorescence microscope (BX51; Olympus, Tokyo, Japan) with 3CCD digital camera ("type":"entrez-nucleotide","attrs":"text":"C77780","term_id":"2518110","term_text":"C77780"C77780; Hamamatsu Photonics, Tokyo, Japan) under ultraviolet excitation. The cultures were harvested every 2 h, and indexes of organelle division were counted. 2.2. Reverse transcriptaseCpolymerase chain reaction Cells were collected by centrifuging at 1400for 3 min and immediately frozen in liquid nitrogen. Nuclear acid isolation buffer (50 mM TrisCHCl, pH7.6, 100 mM EDTA, 300 mM NaCl, 4% SDS, 2% for 5 min at 4C and re-extracted using PCI. Total nucleus acid was precipitated by adding an equal volume of isopropanol and recovered by centrifugation at 15 000for 15 min at 4C. The pellet was melted in DNase I solution (0.1 U/L DNase I, RNase Free; Roshe, 0.4 U/L RNase Inhibitor; Sigma, 10 mM DTT, 10 mM MgCl2) and incubated 1187595-84-1 for 45 min at 37C. Total RNA was precipitated by adding an equal volume 1187595-84-1 of isopropanol and recovered by centrifugation at 15 000for 15 min at 4C. The RNA samples were reverse-transcribed in 20 L of the reaction mix comprising 1 L of Reverse Transcriptase XL (AMV; TaKaRa Bio Inc.), 50 ng/L of oligo(dT) primer (Novagen), 2 U/L of RNase inhibitor and 0.5 mM dNTP mixture (TaKaRa Bio Inc.). The reaction conditions were as follows: 10 min at 25C, 45 min at 42C and 10 min at 70C. Absence of genomic DNA contamination was confirmed by PCR in all the total RNA samples. In the RTCPCR assay, cDNA of and the housekeeping gene were amplified by 22 PCR cycles. The quantity of PCR products was analyzed by electrophoresis in 2% agarose gel. The primers used in PCR were described in Supplementary Table S1. All real-time PCR assay kits were purchased from Applied Biosystems, and utilized according to the manufacturer’s instructions. PCR amplification of each sample was carried out using 7.5 L of Power SYBR Green PCR Master Mix (Applied Biosystems), 5.5 L of distilled water, 0.3 L of.