Project description:In plants, RNA polymerase II (Pol II) transcription of inverted DNA repeats produces hairpin RNAs that are processed by several DICER-LIKE enzymes into siRNAs that are 21-24-nt in length. When targeted to transcriptional regulatory regions, the 24-nt size class can induce RNA-directed DNA methylation (RdDM) and transcriptional gene silencing (TGS). In a forward genetic screen to identify mutants defective in RdDM of a target enhancer leading to TGS of a downstream GFP reporter gene in Arabidopsis thaliana, we recovered a structurally mutated silencer locus, named SM-NM-^T35S, in which the 35S promoter driving transcription of an inverted repeat of target enhancer sequences had been specifically deleted. Although Pol II-dependent, hairpin-derived 21-24-nt siRNAs were no longer generated at the newly created SM-NM-^T35S locus, the GFP reporter gene was nevertheless still partially silenced. Silencing was associated with methylation in a short tandem repeat in the upstream target enhancer and with low levels of 24-nt tandem repeat siRNAs. Introducing an nrpd1 mutation into the SM-NM-^T35S line fully released GFP silencing and eliminated both the tandem repeat methylation and associated 24-nt siRNAs, demonstrating their dependence on Pol IV. Deletion of the 35S promoter thus revealed a Pol IV-dependent pathway of 24-nt siRNA biogenesis that was previously inhibited or masked by the Pol II-dependent pathway in wild-type plants. Both Pol II- and Pol IV-dependent siRNAs accrued predominantly from cytosine (C)-containing segments of the tandem repeat monomer, suggesting that the local base composition influenced siRNA accumulation. Preferential accumulation of siRNAs at C-containing sequences was also observed at an endogenous tandem repeat comprising discrete C-rich and AT-rich sections. Our studies illuminate the potential complexity of siRNA generation at repeat-containing loci and show that Pol IV can act in siRNA biogenesis in the absence of a conventional Pol II promoter. Examination of whole-genome DNA methylation status in transgenic T+S Arabidopsis plant
Project description:A cDNA microarray was constructed from the expressed sequence tags (ESTs) of different developmental stages, and comparative genome hybridization based on microarray procedures were carried out. Dermatophyte species are classified into three genera: Epidermophyton, Microsporum, and Trichophyton. To determine the relationship between these three groups comparative genome hybridization were used in our experiment. Trichophyton rubrum genmic DNA was reference DNA and labelled by Cy3 while the other dermatophytes genomic DNA were test DNA and labelled by CY5. Test and reference DNA were co-hybridized with the T. rubrum cDNA microarray and the numbers of genes shared between each species and T. rubrum were determined. Keywords: Comparative Genomic Hybridization
Project description:A cDNA microarray was constructed from the expressed sequence tags (ESTs) of different developmental stages, and transcriptional profiles of the responses to PHS11A, a fatty acid synthase inhibitor, were determined. The expression profiles of Trichophyton rubrum treated with PHS11A were compared to those of Trichophyton rubrum without drug. Each treatment has three replicates.