Project description:RNA populations in Chlamydomonas reinhardtii Keywords: Highly parallel pyrosequencing Small RNAs were prepared from Chlamydomonas reinhardtii total extracts,ligated to a 3' adaptor and a 5' acceptor sequentially, and then RT-PCR amplified. PCR products were reamplified using a pair of 454 cloning primers and provided to 454 Life Sciences (Branford, CT) for sequencing. For technical details, see Tao Zhao, Guanglin Li, Shijun Mi, Shan Li, Gregory J. Hannon, Xiu-Jie Wang, and Yijun Qi. 2007. A Complex System of Small RNAs in the Unicellular Green Alga Chlamydomonas reinhardtii. Genes & Development
Project description:endogenous small RNAs from Chlamydomonas reinhardtii strain J3(mt-) vegetative cells Keywords: High throughput 454 small RNA sequencing Overall design: Size fractionated small RNA from total RNA extracts was ligated to adapters, purified again and reverse transcribed. After PCR amplification the sample was subjected to 454 high throughput pyrosequencing. Please see www.454.com for details of the sequencing technology.
Project description:BACKGROUND: microRNAs (miRNAs) have been found to play an essential role in the modulation of numerous biological processes in eukaryotes. Chlamydomonas reinhardtii is an ideal model organism for the study of many metabolic processes including responses to sulfur-deprivation. We used a deep sequencing platform to extensively profile and identify changes in the miRNAs expression that occurred under sulfur-replete and sulfur-deprived conditions. The aim of our research was to characterize the differential expression of Chlamydomonas miRNAs under sulfur-deprived conditions, and subsequently, the target genes of miRNA involved in sulfur-deprivation were further predicted and analyzed. RESULTS: By using high-throughput sequencing, we characterized the microRNA transcriptomes under sulphur-replete and sulfur-deprived conditions in Chlamydomonas reinhardtii. We predicted a total of 310 miRNAs which included 85 known miRNAs and 225 novel miRNAs. 13 miRNAs were the specific to the sulfur-deprived conditions. 47 miRNAs showed significantly differential expressions responding to sulfur-deprivation, and most were up-regulated in the small RNA libraries with sulfur-deprivation. Using a web-based integrated system (Web MicroRNAs Designer 3) and combing the former information from a transcriptome of Chlamydomonas reinhardtii, 22 miRNAs and their targets involved in metabolism regulation with sulfur-deprivation were verified. CONCLUSIONS: Our results indicate that sulfur-deprivation may have a significant influence on small RNA expression patterns, and the differential expressions of miRNAs and interactions between miRNA and its targets might further reveal the molecular mechanism responding to sulfur-deprivation in Chlamydomonas reinhardtii.
Project description:Linear tetrapyrrole (bilin)-based phytochrome sensors optimize photosynthetic light capture by mediating massive gene reprogramming in land plants, yet surprisingly, many sequenced chlorophyte (green) algae lack phytochrome genes. Previous studies on the heme oxygenase (hmox1) mutant of Chlamydomonas reinhardtii suggest that bilin biosynthesis in plastids is needed for regulation of a limited nuclear gene network implicated in oxygen detoxification during dark to light transitions. The hmox1 mutant is unable to grow photoautotrophically and poorly acclimates to increased illumination even in the presence of acetate. Here we show that these phenotypes reflect the reduced accumulation of PSI reaction centers as well as a loss of PSI and PSII antennae complexes during photoacclimation. Phenotypically, the hmox1 mutant is similar to the chlorophyll biosynthesis mutants, gun4, crd1 and cth1. However, many of the hmox1 phenotypes can be rescued by the application of exogenous biliverdin IXα, the bilin product of HMOX1; this rescue is independent of photosynthesis but strongly dependent upon blue light. RNA-Seq comparisons of hmox1, 4A+ wild type and two genetically complemented lines also reveal that bilins restore regulation of a small network of photosynthesis-associated nuclear genes. These include genes responsible for chlorophyll biosynthesis (CHLI1/2), PSI light-harvesting (LHCA4) and naphthoquinone metabolism (MEN2), all of which show reduced photoinduction in the hmox1 mutant. We propose that a bilin-based, blue light sensory system is responsible for the maintenance of a functional photosynthetic apparatus in light-grown C. reinhardtii. This critical and possibly ancestral role for bilins may be responsible for retention of bilin biosynthesis in all eukaryotic photosynthetic species. Overall design: We isolated RNA from heterotrophic suspension cultures of 4A+ WT and the hmox1 mutant grown in the presence or absence of 0.1 mM BV IXα before and after transfer to low light.