Astra Bryant<br />Journal Club Presentation<br />Winter 2011<br />
Background:<br />Phosphate, essential for life?<br />
Background:<br />Phosphate, essential for life?<br />1. Isolation of GFAJ-1<br />
Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br />
Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br ...
Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br ...
Background<br />2. <br />1. <br />There are 6 major elements thought to be essential for life: <br />	carbon, hydrogen, ni...
Metabolic pathways confuse Arsenate (AsO43-) and Phosphate (PO43-) </li></ul>3. <br />4. <br />Can arsenate be biochemical...
1. Isolation of GFAJ-1<br />2. <br />1. <br />3. <br />4. <br />Image courtesy of Ed Yong<br />Placed lake sediments into ...
2. Growth in arsenic-rich environment<br />2. <br />1. <br />Media grown in 5 mM AsO43-  plated onto agar dishes containin...
3. Arsenic within GFAJ-1?<br />2. <br />1. <br />Analyzed whole GFAJ-1 cells for arsenic content using Inductively Coupled...
4. Arsenic within DNA?<br />2. <br />1. <br />Gel purified DNA from +As/-P and –As/+P GFAJ-1<br />3. <br />Tested the iden...
Conclusions<br />1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate<br...
Criticism<br />2. Were GFAJ-1 exposed to phosphate concentrations sufficient to support growth?<br />1. Unexplained presen...
Conclusions<br />1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate<br...
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Winter2011 jc

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Winter2011 jc

  1. 1. Astra Bryant<br />Journal Club Presentation<br />Winter 2011<br />
  2. 2.
  3. 3. Background:<br />Phosphate, essential for life?<br />
  4. 4. Background:<br />Phosphate, essential for life?<br />1. Isolation of GFAJ-1<br />
  5. 5. Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br />
  6. 6. Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br />3. Arsenic within GFAJ-1?<br />
  7. 7. Background:<br />Phosphate, essential for life?<br />2. Growth in arsenic-rich environment<br />1. Isolation of GFAJ-1<br />Conclusions:<br />GFAJ-1 incorporates arsenic into DNA, other biomolecules<br />3. Arsenic within GFAJ-1?<br />4. Arsenic within DNA?<br />Criticisms:<br />Dr. Rosie Redfield<br />Dr. Alex Bradley<br />
  8. 8. Background<br />2. <br />1. <br />There are 6 major elements thought to be essential for life: <br /> carbon, hydrogen, nitrogen, oxygen, sulfur and phosphorus.<br />Non-major, “trace” elements can substitute for each other <br /> (e.g. copper for iron as oxygen carriers in arthropods and mollusks.<br /><ul><li>Is elemental substitution possible for the essential elements?</li></ul>Arsenic is a chemical analog of phosphorus<br /><ul><li>Similar atomic radius, electro-negativity
  9. 9. Metabolic pathways confuse Arsenate (AsO43-) and Phosphate (PO43-) </li></ul>3. <br />4. <br />Can arsenate be biochemically substituted for phosphate in an organism specialized for coping with the presence of arsenic?<br />
  10. 10. 1. Isolation of GFAJ-1<br />2. <br />1. <br />3. <br />4. <br />Image courtesy of Ed Yong<br />Placed lake sediments into medium containing increasing AsO43- (100 uM-5mM), trace amounts of PO43- (3.1 uM)<br />In 5 mM AsO43- media shows growth rate of 0.1/day (double every 7 days)<br />
  11. 11. 2. Growth in arsenic-rich environment<br />2. <br />1. <br />Media grown in 5 mM AsO43- plated onto agar dishes containing 40 mM AsO43-, 3.1 uM of PO43- (+As/-P)<br />-As/+P<br />3. <br />4. <br />+As/-P<br />-As/-P<br />GFAJ-1 both increase in number and size in +As/-P conditions<br />-As/+P<br />+As/-P<br />-As/-P<br />
  12. 12. 3. Arsenic within GFAJ-1?<br />2. <br />1. <br />Analyzed whole GFAJ-1 cells for arsenic content using Inductively Coupled Plasma Mass Spectrometry<br />3. <br />4. <br />GFAJ-1 cultured in +As/-P conditions contain larger intracellular amounts of arsenic<br />
  13. 13. 4. Arsenic within DNA?<br />2. <br />1. <br />Gel purified DNA from +As/-P and –As/+P GFAJ-1<br />3. <br />Tested the identities of arsenic’s chemical neighbors using Extended X-ray Absorption Fine Structure<br />4. <br />Tested whether isolated DNA contained arsenic using high-resolution secondary ion mass spectrometry (NanoSIMS)<br />Arsenate is incorporated into DNA backbone<br />Purified GFAJ-1DNA contains arsenic<br />
  14. 14. Conclusions<br />1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate<br />2. GFAJ-1 contain arsenic, localized to protein, metabolite, lipid, and DNA fractions<br />GFAJ-1 can incorporate arsenic into DNA, as well as other biomolecules (NADH, ATP, glucose, acetyl-CoA, other proteins)<br />3. DNA isolated from GFAJ-1 contains arsenic, little phosphate. <br />4. The chemical bond lengths of arsenic are similar to those estimated by models where arsenate replaced phosphate in DNA backbone<br />
  15. 15. Criticism<br />2. Were GFAJ-1 exposed to phosphate concentrations sufficient to support growth?<br />1. Unexplained presence of vacuoles.<br />May contain arsenic, sequestered using membrane pumps implicated in the arsenic resistance of a closely related microbe<br />All solutions contained a background of 3.1 uM PO43-<br />Sargasso Sea contains <10 nM PO43-, contains microbes with phosphate-based DNA<br />4. Arsenic chemical bonds are inherently unstable.<br />3. Assimilation into DNA has not been conclusively demonstrated. <br />Arsenic bonds hydrolyze after ~10 minutes. Isolation of GFAJ-1 DNA involved submersion in water and should therefore have fragmented.<br />How does organism overcome instability?<br />EXAFS primarily measures neighboring molecules, doesn’t show complete incorporation into helical DNA structure<br />Alternate (and classical) method for direct analysis of nucleotides: mass spectrum of DNA sequences<br />
  16. 16. Conclusions<br />1. Bacteria isolated from Mono Lake, CA can grow in conditions with high arsenic and minimal phosphate<br />1. Bacteria isolated from Mono Lake, CA can survive in conditions rich in arsenic and poor in phosphate.<br />2. GFAJ-1 contain arsenic, localized to protein, metabolite, lipid, and DNA fractions<br />2. GFAJ-1 grown in the presence of arsenic will contain intracellular arsenic.<br />GFAJ-1 can incorporate arsenic into DNA, as well as other biomolecules (NADH, ATP, glucose, acetyl-CoA, other proteins)<br />At best, GFAJ-1 can assimilate arsenic into DNA and other biomolecules, perhaps to be used as a functional replacement of phosphate.<br />Alternatively, GFAJ-1 can prioritize phosphate usage while avoiding arsenic poisoning.<br />3. DNA isolated from GFAJ-1 contains arsenic, little phosphate. <br />3. Arsenic may be associated with arsenic-grown DNA, may be a thorough contaminate<br />4. The chemical bond lengths of arsenic are similar to those estimated by models where arsenate replaced phosphate in DNA backbone<br />4. Intracellular arsenic is in the form of arsenate, is not present as ionic species.<br />
  17. 17. Further Reading<br />Dr. Rosie Renfield’s (UBC) criticism (available online, coming soon to Science)<br />Dr. Alex Bradley’s (Harvard) criticism (available at Science Blogs)<br />Carl Zimmer’s article for Slate (with interviews with several eminent scientists)<br />Felicia Wolfe-Simons response to criticism<br />Faculty of 1000 Evaluations<br />Ed Yong’s timeline, from the NASA press release on Dec. 2nd through his post-mortem evaluation on Dec. 10th (available at Discover Magazine blog)<br />Bora Zivkovic’s (PLoS) link dump (available at A Blog Around the Clock)<br />Felicia Wolfe-Simons’ TED Talk on March 2nd. <br />

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