Cycloheximide treatment of cotton ovules alters the abundance of specific classes of mRNAs and generates novel ESTs for microarray expression profiling

Abstract

Fibres of cotton (Gossypium hirsutum L.) are single elongated epidermal cells that start to develop on the outer surface of cotton ovules on the day of anthesis. Little is known about the control of fibre initiation and development. As a first step towards discovering important genes involved in fibre initiation and development using a genomics approach, we report technical advances aimed at reducing redundancy and increasing coverage for anonymous cDNA microarrays in this study. Cotton ovule cDNA libraries (both normalised and un-normalised) from around the time of fibre initial formation have been prepared and partially characterised by sequencing. Re-association-based normalisation partially reduced library redundancy and increased representation of novel sequences. However, another library generated from in vitro cultured cotton ovules treated with the protein synthesis inhibitor, cycloheximide, showed a significantly altered gene representation including a greater proportion of protein phosphorylation genes, transport genes and transcription factors and a much reduced proportion of protein synthesis genes than were identified in the conventional types of libraries. Over 10,000 expressed sequence tag (EST) clones randomly selected from the three libraries were printed on microarray slides and used to assess gene expression in tissue cultured ovules with and without cycloheximide treatment. The microarray results showed that cycloheximide had a dramatic effect in modifying the pattern of the gene expression in cultured ovules, affecting the same types of genes identified in the preliminary analysis on relative EST abundance in the different ovule cDNA libraries. Cycloheximide clearly provided a simple and useful method for enriching novel gene sequences for genomic studies.

Metatranscriptomic Analysis of the Response of River Biofilms to Pharmaceutical Products, Using Anonymous DNA Microarrays

“…Other advantages of biofilms for such studies include their continual exposure to contaminants, abundance, ubiquity, and stationary state (21). Although the subinhibitory pollutant concentrations involved preclude the use of marker genes or species, since subtle and complex effects are expected, an approach quantifying the expression of a range of genes might be appropriate.Anonymous DNA microarrays are frequently used for transcriptomic studies of organisms whose genomes have not been sequenced (5,14,23,26,31,32). Using this approach, cDNA is hybridized to unsequenced DNA or cDNA fragments that are printed on a microarray, and only the probes that display significant responses to the treatment of interest are sequenced.…”

“…Anonymous DNA microarrays are frequently used for transcriptomic studies of organisms whose genomes have not been sequenced (5,14,23,26,31,32). Using this approach, cDNA is hybridized to unsequenced DNA or cDNA fragments that are printed on a microarray, and only the probes that display significant responses to the treatment of interest are sequenced.…”

Cosuppression and RNAi induced by Arabidopsis ortholog gene sequences in tobacco

“…CHX has been shown to increase mRNA stability of several plant genes including the soybean SAUR genes, rice a-amylase gene, alfalfa histone H3 gene, and Arabidopsis MYB2 genes (Franco et al 1990;Gil et al 1994;Sheu et al 1994;Kapros et al 1995;Hoeren et al 1998). In addition, 2.1% (78/3,700) of all the normalized cotton ovule ESTs were up-regulated by CHX treatment (Wu et al 2005). These previous reports imply that a small, but significant, proportion of genes may increase the abundance of their transcripts after translational repression.…”

Genomic approaches to the discovery of promoters for sustained expression in cotton (Gossypium hirsutum L.) under field conditions: expression analysis in transgenic cotton and Arabidopsis of a Rubisco small subunit promoter identified using EST sequence analysis and cDNA microarrays

“…The cDNA clones from the late-season leaf cDNA library were PCR amplified and printed onto CMT-GAPS coated microarray slides (Corning) using a VersArray ChipWriter Pro arrayer (Bio-Rad, Hercules, CA) as described in Wu et al (2005). Post-printing slide processing was performed by baking the slides at 80°C for 3 hours as described in the manufacturer's technical manual.…”

“…Preparation of polyA ϩ RNA, probe labelling with Cy3-dUTP and Cy5-dUTP (Amersham Pharmacia Biotech) slide hybridisation and washing were as described in Wu et al (2005). Microarray images were captured using a GenePix 4000A microarray scanner (Axon Instruments, Union, CA, USA).…”