User:Emmnanuel Quiroz

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Emmanuel Quiroz (an artistic interpretation)
Emmanuel Quiroz (an artistic interpretation)
  • Emmanuel Quiroz
  • Biological Engineering
  • 416 Beacon St
  • Address 2
  • Boston, MA, 02115
  • equiroz AT mit DOT edu

I work in the Wittrup Lab at MIT. I learned about OpenWetWare from Class, and I've joined because To access my classes material.


Chernobyl Fungi Proposal

Topic

Recent studies have discovered fungal species that survive and proliferate in environments of ionizing radiation such as the Chernobyl reactor. These fungi have very efficient DNA repair gene systems that may be homologous to the human pathway. These fungal systems need to be further studied through microarrays and more site-specific anaylsis to a better understanding of how Chernobyl fungi are different. Use of this information can potentially be helpful in cancer research and in developing new therapeutic approaches.

Research Goals

To explore the evolution of the DNA repair mechanism in Chernobyl fungi Cladosporium sphaerospermum to determine differences in gene regulation for potential new approaches in cancer treatment.

  1. Find an efficient DNA repair gene expression in Chernobyl Fungi
  2. Compare the fungi system to that of similar fungi

Research Plan

Perform microarray analysis on Chernobyl fungi( Cladosporium sphaerospermum and Penicillium hirsutum ) and compare their gene expressions of DNA repair systems to the well know genome of yeast (Saccharomyces cerevisiae)

  1. For each species, we will analyze three samples:
    • No treatment: Our overall control
    • Radiation treatment: Simulates conditions found in Chernobyl region (γ-rays)
    • MMS: An alternative DNA-damaging agent, will allow us to determine if the entire DNA pathway has been affected or whether simply radiation resistance genes have been affected
  2. Important considerations:
    • Can we compare three samples on one microarray? (potentially three fluorescent dyes?)
    • How will we compare two different array chips? (new method of log2 analysis required? See References #13 for more analysis ideas.)
    • Do experiment with 50 Gy levels of gamma, reproducing Kimura et al
    • Get sequence to make mircoarray Penicillium roseopurpureum 147 (from contaminated Red Forest soil)
    • fungal isolates and sources in sequence are Penicillium roseopurpureum 147 Red Forest, P. lanosum reactor room, Paecilomyces liliacinus 101 unpolluted soil, P. lilacinus 1941 Red Forest, Penicillium hirsutum 3 hot particles, Cladosporium sphaerospermum 60 reactor room, C. cladosporioides unpolluted soil, C. sphaerospermum 3176 unpolluted soil, and C. cladosporioides 10 reactor room).
  3. Past experimtents to relate to
    • Kimura et al analyzed S. cerevisiae using mircroarrays and found: Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and c-rays). Similarly, for X- and c-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. [12]

BLAST two species' sequences against each other

  1. Compare differences in the sequences to microarray analysis result
  2. This will help confirm that variations in microarray fluorescence accurately correlated to differences in the regulatory pathway or genetic sequence of the two fungi
  3. Taxonomy NCBI taxonomy database
    • Saccharomyces cerevisiae: Eukaryota; Fungi; Dikarya; Ascomycota; Saccharomycotina; Saccharomycetes; Saccharomycetales; Saccharomycetaceae; Saccharomyces
    • Cladosporium sphaerospermum: Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Dothideomycetes; Dothideomycetidae; Capnodiales; Davidiellaceae; mitosporic Davidiellaceae; Cladosporium
    • Penicillium hirsutum: Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Eurotiomycetes; Eurotiomycetidae; Eurotiales; Trichocomaceae; mitosporic Trichocomaceae; Penicillium
    • Penicillium chrysogenum: Eukaryota; Fungi; Dikarya; Ascomycota; Pezizomycotina; Eurotiomycetes; Eurotiomycetidae; Eurotiales; Trichocomaceae; mitosporic Trichocomaceae; Penicillium; Penicillium chrysogenum complex
  4. Relative to Penicillium h.: Penicillium c. sequenced and is used to produce penicillin

Conduct more site-specific analysis to more intimately determine changes in one particular gene (See possible gene candidates below)

  1. Either use "pre-determined" gene involved in DNA repair that we have explored in a paper (See References) or a gene found via our microarray
    • Experiment Ideas:
    • Potentially create a knockout of the gene and see how repair/cell cycle is affected
    • Apply some sort of agent to up- or down-regulate the gene

Potential Gene Targets

  1. repair (RAD50, RAD51)
  2. recombination (HRP1)
  3. chromosome stability (CHL1, CTF4)
  4. endocytosis (VID21)
  5. ubiquitin degradation (GRR1)
  6. transcription (BUR2)

References

  1. Ionizing radiation: how fungi cope, adapt, and exploit with the help of melanin ----- Fungi are seen to proliferate in environments of ionizing radiation such as the Chernobyl reactor and on the outer shell of space craft. These fungi show an increase in melanin expression which suggest that melanin can function in energy capture and prevent DNA damage. It was also seen through microarray analysis that exposure to radiation caused an up regulation in DNA repair genes. Interestingly, many of the radiation resistance genes share significant homology with human genes that might be exploited in the development of novel radiation-based cancer therapies. Link
  2. Fungi and Ionizing Radiation from Radionuclides ----- Radionuclides, atoms with unstable nuclei due to excess energy that undergo radioactive decay, have provided interesting clues as to gene regulation since the Chernobyl power plant accident. Researchers have focused on various forms of fungi as a model of these effects because its large surface area allow for greater uptake capacity of nutrients and make it preferential to surround host plants. Through broad microarray analysis of various fungal stains, short term exposure of yeast to gamma and X radiation has been shown to elicit the up-regulation of genes involved in DNA cell repair, cell rescue defense, and cell fate and metabolism. Cladosporium cladosporioides and Penicillium roseopurpureum (saprotrophic micro-fungi) in particular were shown to over grow on carbon-based radioactive debris, and while radionuclides were incorporated into the cytoplasm, they were highly absorbed in ion-exchange sites (i.e. the cell membrane). Additionally, intense radiation of soil fungal communities led to a dominance of melanized fungal species, indicating a potential radiological protective mechanism in the pigment. Dighton, John; FEMS Microbiol Lett 2008 Vol. 281 Iss. 2 p. 109 - 20ppublish Link
  3. Chernobyl effect: growth characteristics of soil fungi Cladosporium cladosporioides (Fresen) de Vries with and without positive radiotropism -- Zhdanova, N Fomina, M Redchitz, T Olsson, S Polish Journal of Ecology [Pol. J. Ecol.]. Vol. 49 2001 Vol. 49 Iss. 4 p. 309 - 318 Link
  4. Fungi from Chernobyl: mycobiota of the inner regions of the containment structures of the damaged nuclear reactor Zhdanova, NN Zakharchenko, VA Vember, VV Nakonechnaya, LT Mycological Research [Mycol. Res.]. Vol. 104 2000 Vol. 104 Iss. 12 p. 1421 - 1426 Link
  5. Normal expression of DNA repair proteins, hMre11, Rad50 and Rad51 but protracted formation of Rad50 containing foci in X-irradiated skin fibroblasts from radiosensitive cancer patients (RAD51 in cancer cells) C Djuzenova, B Mühl, R Schakowski, U Oppitz and M Flentje British Journal of Cancer (2004) 90, 2356–2363 Link
  6. Alternative recombination pathways in UV-irradiated XP variant cells (XP variant HR pathway) Charles L Limoli1, E Giedzinski1 and J E Cleaver. Oncogene (2005) 24, 3708–3714. doi:10.1038/sj.onc.1208515 Published online 7 March 2005 Link
  7. RECOMBINATION PROTEINS IN YEAST (helpful detailed descriptions of Rad50 and many other proteins) Berit Olsen Krogh and Lorraine S. Symington. Annual Review of Genetics Vol. 38: 233-271 (December 2004). Link
  8. Genes required for ionizing radiation resistance in yeast Craig B. Bennett, L. Kevin Lewis, Gopalakrishnan Karthikeyan, Kirill S. Lobachev, Yong H. Jin, Joan F. Sterling, Joyce R. Snipe & Michael A. Resnick. Nature Genetics 29, 426 - 434 (2001) Published online: 19 November 2001. Link
  9. Functional genomics of the yeast DNA-damage Gerard Cagney, David Alvaro, Robert JD Reid, Peter H Thorpe, Rodney Rothstein and Nevan J Krogan, Genome Biology 2006, 7:233 Link
  10. Multiple-laboratory comparison of microarray platforms (Methods of Microarray Comparative Analysis) Rafael A Irizarry, Daniel Warren, Forrest Spencer, Irene F Kim, Shyam Biswal, Bryan C Frank, Edward Gabrielson, Joe G N Garcia, Joel Geoghegan, Gregory Germino, Constance Griffin, Sara C Hilmer, Eric Hoffman, Anne E Jedlicka, Ernest Kawasaki, Francisco Martínez-Murillo, Laura Morsberger, Hannah Lee, David Petersen, John Quackenbush, Alan Scott, Michael Wilson, Yanqin Yang, Shui Qing Ye & Wayne Yu. Nature Methods 2, 345 - 350 (2005) Published online: 21 April 2005. Link
  11. Interactions of Fungi and Radionuclides in Soil John Dighton, Tatyana Tugay, and Nelli Zhdanova Soil Biology ISSN 1613-3382 Volume 13 Book Microbiology of Extreme Soils Link
  12. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and γ-rays Shinzo Kimura, Emi Ishidou, Sakiko Kurita, Yoshiteru Suzuki, Junko Shibato, Randeep Rakwal, Hitoshi Iwahashi Biochemical and Biophysical Research Communications Volume 346, Issue 1, 21 July 2006, Pages 51-60 Link
  13. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum Nature Biotechnology 26, 1161 - 1168 (2008) Published online: 28 September 2008 Link
  14. MIPS Saccharomyces cerevisiae genome database
  15. Agilent SurePrint Technology
  16. Agilent Technologies Custom eArray
  17. [http://ec.asm.org/cgi/content/full/2/6/1253 Potential Microarray Protocol
  18. MycoBank

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