Project description:The molecular machine that synthesizes RNA in Eucarya and Archaea, RNA polymerase, is composed of 11 or 12 subunits M-^V 9 or 10 that form the core holoenzyme, and a heterodimer formed from subunits E and F that associates with the core.<br><br>In this study we used a recombinant archaeal MbRpoE/F heterodimer to capture cellular mRNA and a custom Agilent microarray to determine which mRNA it binds. Transcripts bound by the heterodimer were identified through competitive hybridization of the total RNA obtained from Methanococcoides burtonii and the RNA obtained through the selection of the transcripts that interact with the MbRpoE/F heterodimer bound to the column.
Project description:Methanococcoides burtonii is member of the Archaea that is a valuable model for studying cold adaptation. We developed a Agilent microarray for determing which genes are expressed in operons, and which are differentially expressed at low (4°C) or high (23°C) temperature. Agilent 8 x 15K custom gene expression microarrays containing 15128 probes were designed based on the M. burtonii genome sequence. The Microarrays were constructed using 60-mer oligonucleotides covering 2236 genes (86.7% of the total number) on the coding strand (10153 oligonucleotides) and the complementary strand (3671 oligonucleotides), and a large number of intergenic regions (707 oligonucleotides) (Table 1). Each gene and intergenic region was covered by 1 to 6 oligonucleotides (average of 4 per gene). Eight independent replicates were performed using competitive hybridization comparing 8 cultures grown at 4°C vs 8 grown at 23°C. Due to the fact that different ORFs have different numbers of oligonucleotides (ranging from one to six) and that experiments were performed in 8 different replicates, each gene (or intergenic region) has 8 48 measures of fluorescence.
