In vivo NMR as a tool for probing molecular structure and dynamics in intact Chlamydomonas reinhardtii cells

GO biological process enrichment for the 882 deletion strains below the threshold of detection by microarray in the BCprot relative to the gene universe of strains present in at least one deletion collectio

Essential Genes and Protein Complex Subunits Minimize Noise in Expression

<p>Binning analysis of (A) essential genes and (B) protein complex subunits. All genes for which transcription and translation rate data were available were separated into 15 bins by their protein production rate. Each bin was then separated into thirds by number of translations per mRNA. The two-thirds in each bin with the most extreme transcription and translation are shown: black bars are the number of each type of gene (essential or complex subunit) in the third of each bin with the lowest number of translations per mRNA and the highest transcription rate, and thus low noise; gray bars are the number of each type of gene in the third with the highest number of translations per mRNA and the lowest transcripton rate, and thus high noise. Bins are ordered by their rate of protein synthesis. The number of asterisks indicates the Fisher's exact test probability of observing the values for each bin under the null model of independence. *, <i>p</i> ≤ 0.02; **, <i>p</i> < 0.005; ***, <i>p</i> < 0.0005.</p

Haloferax transcripts

Manually-curated transcripts based on RNA-seq dat

Control MNase-seq data aligned to Haloferax genome (BAI file)

Control MNase-seq data aligned to Haloferax genome (BAI file

Workflow for methotrexate resistance screen.

<p>The plasmid-borne <i>dfr1</i> variomics library contains ~2 x 10⁵ independent <i>dfr1</i> alleles maintained in a diploid <i>DFR1</i>/<i>dfr1</i>Δ heterozygote strain. The diploid pool was sporulated and cultured in haploid selective media to generate a <i>dfr1</i> pool in a <i>dfr1</i>Δ haploid background. Both diploid and haploid strains were grown in the presence of MTX (2 mM) or DMSO solvent control (2% v/v) over a 6-day timecourse (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006275#pgen.1006275.s002" target="_blank">S2 Fig</a>). MTX-treated cultures were subsequently harvested for plasmid extraction followed by PCR amplification of the dfr1 alleles. Nextera XT libraries were prepared for DFR1-amplicon sequencing, and non-reference <i>dfr1</i> variant alleles were identified using the RVD analysis tool. Candidate <i>dfr1</i> point mutations were validated by constructing the individual <i>dfr1</i> mutants and their MTX resistance confirmed in growth assays.</p

Validation of methotrexate resistant dfr1 mutations.

<p>The average fitness of the candidate <i>dfr1</i> haploid (A) and diploid (B) mutants upon exposure to MTX (1 mM sublethal dose) or DMSO solvent (2% v/v) were evaluated over 24 hours in a Tecan shaker-reader at 30°C. Mutant alleles expressed in the presence of a wild-type <i>DFR1</i> copy are listed with (+). Mutants are colored according to their relative doubling time in comparison to the wild-type strains: BY4742 (WT, A) and <i>DFR1</i>/<i>dfr1</i>Δ (WT, B) with: blue for longer doubling time (Slower than WT), grey for comparable doubling time (Fit as WT), and red for shorter doubling time (Faster than WT). The average growth under MTX conditions of the control strains are indicated with a dashed line. Error bars indicate standard error, n = 3.</p

Nucleosome MNase-seq data aligned to Haloferax genome (BAM file)

Aligned nucleosome read

Control MNase-seq data aligned to Haloferax genome (BAM file PART2)

naked DNA control (MNase-seq

Mapping of the <i>dfr1</i> mutations onto the yeast DFR1 model.

<p>(A) Cartoon representation of the structural model for the yeast DFR1, colored by sequence conservation (red is conserved, blue is divergent), with surface shown in transparency. The identified resistance point mutations are indicated with a sphere. MTX is highlighted in yellow, and NADPH in green. Mutations largely cluster around conserved residues near the active site of the enzyme. The <i>dfr1</i> mutations of residues interacting with MTX (yellow, B) or NADPH (green, C) are mapped onto the yeast DFR1 model. The represented protein is colored by sequence conservation as in (A).</p

Control MNase-seq data aligned to Haloferax genome (BAM file PART1)

naked DNA control (MNase-seq