Detecting Anthracycline Induced Intrachromosomal Recombination in Saccharomyces cerevisiae Using a D

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Detecting Anthracycline Induced Intrachromosomal
Recombination in Saccharomyces cerevisiae Using a
Del Assay

• Jafa Armagost
• Alexis Snow
• Alpha Chi
• Dr William Mackay
• Edinboro University of Pennsylvania

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Procedure for Del Assay

• 1. A single RS112 colony was incubated in 5 ml
  SC-LEU medium for 24 hr at 30o C
• 5 µl of the culture was exposed to daunomycin,
  adriamycin or MMS and grown at 30o C for 17 hr
• Exposed culture was plated on SC-HIS for 2 days
  at 30o C
• 4. Colonies were counted

Schiestl RH, Gietz RD, Mehta RD, and Hastings PJ
Carcinogenesis 1989 10(8)1445-1455
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Intrachromosomal Recombination
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Future Research

• Mouse in vitro Del Assay
• The mouse p gene codes for a melanosomal
  integral membrane protein that is required for
  the black coat color of mice. Mice with a
  recessive homozygous pink eyed unstable (pun)
  mutation shows a dilution in skin and eye
  pigmentation. The pun mice will be injected with
  0.2 ml (120 mg/kg) of a anthracycline-saline
  solution 10.5 days after conception. Mice will be
  examined 13 days after birth for induced spotting
  showing induced reversion events.

Schiestl RH, Aubrecht J, Khogali F, Carls N.
Proc. Natl. Acad. Sci. 1997 94 (9) 45764581
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Abstract

• For more than thirty years, daunomycin has
  represented one of the most commonly used classes
  of anticancer drugs. Studies in our lab have
  shown that daunomycin can induce mutations in the
  bacterium Salmonella typhimurium. The objective
  of this study is to use a deletion (Del) assay to
  test the role of DNA repair systems in the
  recognition and removal of mutagenic
  daunomycin-induced DNA adducts by analyzing a DNA
  recombination system in the yeast Saccharomyces
  cerevisiae.

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Materials and Methods

• Strain RS112
• HIS3 pRS6/his3-?200 (his33'-LEU2-
  pBR322-his35')
• Mata/a ura3-52/ura3-52
• leu2-3,112/leu2-?98 trp5-27/TRP5
• arg4-3/ARG4 ade2-40/ade2-101
• ilv-92/ILV1 LYS2/lys2-801

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Bibliography

• Mackay WJ, Phelps LA, Cauchi, AA, and Weaver LT.
  The mutagenic effects of anthracyclines in the
  bacterium Salmonella typhimurium Induction of
  transition mutations with daunomycin. 2000 Texas
  J. Sci. 52(3) 223-229
• 2. Schiestl RH, Aubrecht J, Khogali F, Carls N.
  Carcinogens induce reversion of the mouse
  pink-eyed unstable mutation. Proc. Natl. Acad.
  Sci. 1997 94 (9) 45764581

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Results of Ames Assay with Daunomycin (120 mg/ml)
Strain
Induction Fold
44.6
TA 98
7.6
TA 7004 (GC AT)
7.3
TA 7001 (AT GC)
c2 p Mackay WJ, Phelps LA, Cauchi, AA, and Weaver LT
Texas Journal of Science 52 (3) 223-229 (August
2000)
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• Chemical
• Daunomycin
• 170 mg/ml
• 340 mg/ml
• Methyl Methanesulfonate (positive control)
• 130 mg/ml
• 65 mg/ml
• Media
• SC-LEU
• selects for Leu prototrophs
• SC-HIS
• selects for His prototrophs

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Results of Del Assay
Chemicals Mutation frequency Daunomycin 170
mg/ml 1 x 10-3 Daunomycin 340 mg/ml 3 x
10-3 MMS 130 mg/ml 1 x 10-1 MMS 65 mg/ml
2 x 10-2 Uninduced 1 x 10-4
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• 3. Schiestl RH, Gietz RD, Mehta RD, and Hastings
  PJ. Carcinogens Induce Intrachromosomal
  Recombination in Yeast. Carcinogenesis 1989
  10(8)1445-1455
• 4. Sommers CH, Mackay WJ, Nalezny J, Gee P,
  Benjamin M, and Farr SB. Induction of DEL
  Recombination in the Yeast Saccharomyces
  cerevisiae Using a Microtiter Plate Assay Format.
  In Vitro Tox. 1995 8(1) 37-47