Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Alexandrium_copepod_interaction_II

ABSTRACT: A. tamarense strains were incubated in 500mL glass bottles at a concentration of approximately 5M-W106 cells L?1 in K/10 medium. Grazed treatments received 10 Centropages typicus individuals per bottle.

ORGANISM(S): Alexandrium tamarense

SUBMITTER: Sylke Wohlrab

PROVIDER: E-MEXP-3422 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The screen for genes involved in the spontaneous loss of heterozygosity mutagenesis (SLM) in diploid cells was done on the collections of deletion strains of Saccharomyces cerevisiae created by the Saccharomyces Genome Deletion Project (http://www-sequence.stanford.edu/group/yeast_deletion_project/). Screens were performed on the pools of clones. Barcode microarrays were used to detect the increased SLM frequency leading to relative increase in abundance of deletion clone in a pool. Collections used were: Homozygous Diploid (HD) and Essential (E) mixed to create single HDE pool. Three mutagenesis markers were used: (1) naturally existing mating type locus, and two newly introduced markers, (2) CAN1/can1delta, (3) URA3/ura3delta. 1. To detect the increased SLM frequency at the mating type locus HDE pool was crossed to MATa or MATalpha strain. Deletion clones that gained mating competence due to loss of heterozygosity (LOH) at the mating type locus gave rise to clones with the ability to grow on minimal medium. The relative abundance of these clones was compared to the abundance in the original HDE pool. 2. To detect the increased SLM frequency at the CAN1/can1delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of CAN1 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under canavanine selection and could be estimated by comparing it to the abundance when pool was grown without selection. 3. To detect the increased SLM frequency at the URA3/ura3delta locus the derivative HDE pool was created. Every cell of this pool had one of two copies of URA3 gene deleted. The increased SLM frequency led to increased relative abundance of respective deletion clone under 5M-^R-fluoroorotic acid selection and could be estimated by comparing it to the abundance when pool was grown without selection. 4. For screens of SLM at CAN1 and URA3 markers the resistance control experiments were performed to detect genes whose deletion is sufficient to enable yeast cells with functional CAN1 or URA3 to grow in the presence of canavanine or 5M-^R- fluoroorotic acid respectively. 5. To include in the analysis slow-growing deletion clones control hybridizations were performed to detect genes whose deletion extends the doubling time of yeast cells.

Project description:RNA pull-down assay. For the recombinant protein pull-down assays, 50 M-5g of recombinant His-tag TcRBP40 protein were bound to 100 uL of Ni-NTA resin (Qiagen) overnight at 4M-0C. 100 M-5g of total RNA from epimastigotes were incubated with the bound protein in 500 M-5l EMSA buffer at 4M-0C for 2 h, in the presence of Heparine and Spermidine as competitors. Bounded and supernatant samples were separated. The bound sample was washed with the same buffer three times, soft-mixing for 10 min each. After washing, RNA present in the bound and supernatantM- fractions were purified. RNA purification and amplification: RNA was extracted using the RNeasy mini kit (Qiagen). Linearly amplified RNA (aRNA) was generated with the MessageAmpM-^YII aRNA Amplification kit (Ambion), according to the manufacturerM-^Rs manual. Microarray analysis: The microarray was constructed with 70-mer oligonucleotides. Due to the hybrid and repetitive nature of the sequenced T. cruzi strain, all coding regions (CDS) identified in the genome (version 3) were retrieved and clustered by the BLASTClust program, using parameters of 40% coverage and 75% identity. For probe design, it was used ArrayOligoSelector software (v. 3.8.1), with a parameter of 50% G+C content. Was obtained 10,359 probes for the longest T. cruzi CDS of each cluster, 393 probes corresponded to the genes of an external group (Cryptosporidium hominis) and 64 spots contained only spotting solution (SSC 3x), given 10,816 spots in total. These oligonucleotides were spotted from a 50 M-5M solution onto poly-L-lysine coated slides and cross-linked with 600 mJ UV. Each probe corresponding to the T. cruzi genes was identified according to the T. cruzi Genome Consortium annotation (www.genedb.org). We compared bound and unbound mRNA, extracted from two independent pull-down assays, in a dye-swap design including four slides. Microarray images were analyzed by Spot software (Spot). The Limma package (Smyth GK, 2004) was used for background correction by the normexp method, intra-slide normalization by the printtiploess method and inter-slide normalization by the quantile method. The results for the two intra-slide probe replicates were then averaged. The pull-down results were averaged, and probes displaying more than a two-fold difference between the bound and unbound fractions were selected, at FDR 1%.

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Alexandrium_copepod_interaction_II

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