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.
Project description:Proliferating cell nuclear antigen (PCNA) is a DNA-clamping protein that is responsible for increasing the processivity of the replicative polymerases during DNA replication and repair. The PCNA from the eurypsychrophilic archaeon Methanococcoides burtonii DSM 6242 (MbPCNA) has been targeted for protein structural studies. A recombinant expression system has been created that overproduces MbPCNA with an N-terminal hexahistidine affinity tag in Escherichia coli. As a result, recombinant MbPCNA with a molecular mass of 28.3 kDa has been purified to at least 95% homogeneity and crystallized by vapor-diffusion equilibration. Preliminary X-ray analysis revealed a trigonal hexagonal R3 space group, with unit-cell parameters a = b = 102.5, c = 97.5 angstrom. A singleMbPCNA crystal was subjected to complete diffraction data-set collection using synchrotron radiation and reflections were measured to 2.40 angstrom resolution. The diffraction data were of suitable quality for indexing and scaling and an unrefined molecular-replacement solution has been obtained.
Project description:Hyphopichia burtonii has much stronger osmotolerance than model Saccharomyces cerevisiae and other yeast. The transcriptomic changes by RNA-seq analysis in several hyperosmotic stresses and YPD condition as control were investigated, expecting changes in the expression patterns of genes that do not exist or change in other yeast.
Project description:In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry. Accordingly, traditional efforts to enhance Rubisco catalysis using protracted "trial and error" protein engineering approaches have met with limited success. Here we demonstrate the versatility of high throughput directed (laboratory) protein evolution for improving the carboxylation properties of a non-photosynthetic Rubisco from the archaea Methanococcoides burtonii. Using chloroplast transformation in the model plant Nicotiana tabacum (tobacco) we confirm the improved forms of M. burtonii Rubisco increased photosynthesis and growth relative to tobacco controls producing wild-type M. burtonii Rubisco. Our findings indicate continued directed evolution of archaeal Rubisco offers new potential for enhancing leaf photosynthesis and plant growth.
