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Absence of Detectable Arsenate in DNA from Arsenate-Grown GFAJ-1 Cells

Science  27 Jul 2012:
Vol. 337, Issue 6093, pp. 470-473
DOI: 10.1126/science.1219861

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  1. Fig. 1

    Growth curves of GFAJ-1 in AML60 medium supplemented with different concentrations of phosphate. Each line is the mean of 10 replicate 300-μl cultures in wells of a Bioscreen C Growth Analyzer. The phosphate additions used to replicate the “–P” and “+P” conditions of (1) are indicated.

  2. Fig. 2

    Integrity of GFAJ-1 chromosomal DNA after long-term storage. Lanes: 1 and 7, Hind III digest of lambda DNA; 2, Haemophilus influenzae chromosomal DNA; 3 to 6, GFAJ-1 chromosomal DNA grown in the specified combinations of As and P (–As: no arsenate; +As, 40 mM arsenate; –P, 3 μM added phosphate; +P, 1500 μM added phosphate). (A) About 100 ng of GFAJ-1 DNA immediately after purification. (B) The same DNAs (200 ng/lane) after 2 months of storage in tris-EDTA at 4°C. (C) The same DNAs as in (B), but 800 ng/lane and after 10 min at 95°C.

  3. Fig. 3

    LC-MS analysis of arsenate in purified and CsCl-fractioned DNA from arsenate-grown GFAJ-1 cells. Representative extracted ion chromatograms for arsenate [m/z = 140.9174 ± 3 parts per million (ppm)] are shown as the chromatographic retention time (in minutes) plotted against intensity (in ion counts). Sample identity is indicated to the right, along the axis extending into the page. DNA from arsenate-grown GFAJ-1 cells (+As/–P undigested gDNA) was analyzed by LC-MS at a 1:10 dilution, as were the water wash (+As/–P wash of gDNA), the same DNA after washing and enzymatic digestion (+As/–P washed, digested DNA), and finally, fractions of the same DNA after a CsCl gradient purification and digestion (+As/–P CsCl fractions #1 to #8, with DNA concentrating in fractions #6, #7, and #8). Potassium arsenate standards (Std 1.7e-6 to 1.7e-8 [M]) and a water blank were also analyzed. One of four representative experiments is shown.

  4. Fig. 4

    LC-MS analysis of deoxynucleotides from purified and CsCl-fractioned DNA from arsenate-grown GFAJ-1 cells. Representative extracted ion chromatograms are shown as the chromatographic retention time (in minutes) plotted against intensity (in ion counts). One of four representative experiments is shown. (A and B) Extracted ion chromatograms for (A) deoxyadenosine-phosphate (dAMP; m/z = 330.0609 ± 5 ppm) and (B) its arsenate analog deoxyadenosine-arsenate (dAMA; m/z = 374.0087 ± 5 ppm). DNA from arsenate-grown GFAJ-1 cells (+As/–P washed, digested DNA) was washed, digested, and analyzed by LC-MS, as was the same DNA after a CsCl gradient purification and digestion (+As/–P CsCl fractions #1 to #8). To keep the peak on scale, the signal for +As/–P washed, digested DNA has been multiplied by 0.5. This observed large peak matches the known retention time of dAMP. (C and D) Extracted ion chromatograms for (C) the dideoxynucleotide deoxyadenosine-phosphate (dAMP-dAMP; m/z = 643.1185 ± 5 ppm) and (D) its mono-arsenate analog deoxyadenosine-arsenate–deoxyadenosine-phosphate (dAMA-dAMP; m/z = 687.0663 ± 5 ppm). DNA from arsenate-grown GFAJ-1 cells (+As/–P washed, digested DNA) was washed, digested, and analyzed by LC-MS, as was the same DNA after a CsCl gradient purification and digestion (+As/–P CsCl fractions #1 to #8). Partially digested –As/+P DNA shows a large peak at the exact mass of dAMP-dAMP.